This invention relates to the production of antibodies directed against exotoxin A from Pseudomonas aeruginosa.
Pseudomonas aeruginosa is a highly virulent pathogen which infects patients receiving immunosuppressive therapy or suffering from severe thermal burns or other serious injuries, cystic fibrosis, or neoplastic diseases. Mortality from P. aeruginosa has been reduced as the result of such therapeutic agents as mafenide acetate and silver salts which inhibit bacterial colonization of the burn wound surface, potent antibiotics for treating bacteremia, and barrier isolation to minimize contact of the patient with hospital flora. Such agents, however, have only proved partially successful in controlling the morbidity and mortality associated with Pseudomonas infections.
Recently, researchers have found that specific antibodies constitute a critical immunologic defense mechanism against Pseudomonas disease; therefore, vaccines have been administered to patients in attempts to increase antibody titers in the patients. No non-toxic vaccines have been found to date which are particularly effective against the pathogen.
It is not yet clear what components of P. aeruginosa are responsible for its virulence. Many different types of infections are recognized, from acute localized eye infections and chronic lung infections to generalized systemic infections and septicemia. One of the most extensively studied components of P. aeruginosa is exotoxin A. (Iglewski et al., Methods Enzymol., 60, 780-793 (1979)). Exotoxin A is on a weight basis the most potent extracellular product of P. aeruginosa and is produced by about 90% of clinical isolates regardless of Fisher-Devlin-Gnabasik immunotype. The mechanism of action of exotoxin A is similar to that of diphtheria toxin in that exotoxin A is synthesized as a proenzyne which catalyzes the transfer of the ADP ribose moiety from NAD into covalent linkage with elongation factor 2, thereby potentially inhibiting mammalian protein synthesis.
Antibody to endotoxin A has been found in normal human adults and was studies by Cross et al., J. Inf. Dis., 142, 538 (1980) and by Pavlovskis et a., Inf. Immun., 18, 596-602 (1977). See also Pollack, Rev. Inf. Dis., 5, 979-984 (1983). Pollack, J. Inf. Dis. 147, 1090 (1983) studied the immunologic reactivity and opsonic and protective activity against Pseudomonas aeruginosa of twenty-seven lots of human immune globulin from seven producers. All contained hemagglutinating antibodies to exotoxin A.
Cryz et al., Inf. and Imm., 40, 659-664 (1983) indicate that immunoglobulin G (IgG) fractions directed against exotoxin A are protective in experimentally infected animals. Hancock et al., Inf. and Imm., 37, 166-171 (1982) describe preparation of mouse monoclonal antibodies against P. aeruginosa outer membrane antigens. Galloway et al., Inf. and Imm., 44, 262-267 (1984) described mouse monoclonal antibodies specific to extoxin A. The literature reports the preparation of human monoclonal antibodies directed against various antigens. EP No. 81303286.9 published Jan. 27, 1982 is directed to human monoclonal antibodies and suggests making antibodies to "pathogen surface antigens" and "toxins". In addition, Foung et al., J. Immun. Meth., 70, 83-90 (1984) discloses human monoclonal antibodies production from an EBV-transformed B cell line by fusion to a human-mouse hybridoma. Several fushion partners are described by D. Buck et al., Chapter 11 of Monoclonal Antibodies and Functional Cell Lines, ed. by R. Kennett et al., Plenum Publishing Corp., 1984. Mutharia et al., Inf. and Imm., 45, 631-636 (1984) describe monoclonal antibodies specific for E. coli J5 LPS. Europ. Pat. Publications 107,528 and 105,804 describe cell lines capable of producing human monoclonal antibodies against a bacterial toxin. In addition, GB Nos. 2,086,937; 2,113,715; EP Nos. 57,107; 62,409; 118,893; 124,301 and 131,878 all relate to manufacture of human monoclonal antibodies from hybrid cells.
There is a need to develop monoclonal antibodies for passive immunotherapy of patients infected with P. aeruginosa.